Membrane having high affinity for low density lipoprotein-cholesterol from whole blood

ABSTRACT

The present invention relates to the efficient removal of low density lipoprotein cholesterol complex (LDL-C) from whole blood. More specifically, it relates to the use of an immobilized affinity agent on a microporous plasmapheresis membrane. The immobilized affinity agent is polyacrylic acid bound directly and/or through an interaction with silica and/or calcium chloride to a microporous hollow fiber membrane.

TECHNICAL FIELD

The present invention relates to the efficient removal of low densitylipoprotein cholesterol complex (LDL-C) from whole blood. Morespecifically, it relates to the use of an immobilized affinity agent ona microporous plasmapheresis membrane. The immobilized affinity agent ispolyacrylic acid bound directly and/or through an interaction withamorphous silica and/or calcium chloride to a microporous hollow fibermembrane.

BACKGROUND

Atherosclerosis is the thickening and loss of elasticity in the innerwalls of arteries, accompanied by the formation of small fatty moduleson the artery walls and degeneration of the affected area.Atherosclerosis presented in the form of coronary heart disease andcerebrovascular diseases are major causes of morbidity and mortality inmany industrial countries. Elevated plasma levels of low densitylipoprotein-cholesterol complex (LDL-C) correlate with an increased riskfor the development of atherosclerosis.

Patients at high risk for atherosclerosis are encouraged to make dietarychanges in an attempt to control LDL-C levels. However, patientcompliance is not always high and there is a large patient populationwhich cannot control LDL-C levels merely through dietary modifications.

Drug therapy is also commonly used to try to lower LDL-C levels. Whiledrug therapy is effective for many are resistant to drug therapy or whosuffer too many side effects to warrant its use.

In addition to dietary changes and drug therapy, attempts have been madeto remove LDL-C directly from the plasma of patients throughextracorporeal methods. These methods include plasma exchange,filtration based on molecular size, immunoadsorption, the paringprecipitation and dextran sulfate adsorption. While these methodseffectively remove LDL-C from plasma, they also remove varyingquantities of desirable plasma components. The plasma exchange methodremoves all plasma and replaces the volume with plasma or albuminreplacement solutions. All valuable plasma components, such as highdensity lipoprotein (HDL), and proteins are removed in addition to theLDL-C. The other methods, while better than plasma exchange, havevarying degrees of specificity for only LDL-C. With filtration based onmolecular size, there is considerable loss of proteins with molecularweights greater than 250-400 kD. Immunoabsorption is specific for LDL-Conly, but its efficiency for removal of LDL-C is not as great as othermethods. Heparin precipitation and dextran sulfate adsorption removeLDL-C, but a loss of 20-40% of HDL is generally expected; also theadsorbing capacities are fairly low. Since HDL plays an important rolein reducing a patient's risk for atherosclerosis, a method whicheliminates or minimizes the loss of HDL is highly desirable.

Previous filtration methods have also utilized carriers, such as agarosebeads, which lack mechanical strength, and as a result are difficult tohandle and operate. When fluid is passed through these carriers, thereis a high probability of blockage. Additionally, these carriers may bedestroyed by sterilization techniques. These carriers might also leachmaterials into the patient fluid.

Polyacrylate has been tested as a sorbent for lipoproteins from humanplasma (Thies et al., Artificial Organs (1988) 12(4):320-324).Negligible loss of HDL and plasma proteins was shown with thisabsorbent. Polyacrylate has been attached to cellulosic beads throughamide linkages. While the preparation was useful, it was not optimal forthe treatment of whole blood. As mentioned previously, cellulosic beadsdo not have good mechanical strength, block easily, and are not easilysterilized.

Kuroda et al. (EP 0143369) describe a porous adsorbent for absorbing lowdensity lipoproteins having a silanol group and a synthetic polyanionlinked with the surface. To prevent clogging, the porosity of theadsorbent must be distributed over a broad diameter range. By contrast,the microporous membrane of the present invention has uniform porediameters. Murakami (Japanese P.A. 01-229878) describes porous polyesterfibers coated with methacrylic acid which are useful to remove bilirubinor LDL from body fluids. Sterilization of polyester fibers can beproblematic. Kuroda et al. (Japanese P.A. 63-232845) describe anabsorbent material having on its surface a synthetic linear polymerwhich has both a carboxyl group and sulfate or sulfonate groups.

To date, the majority of extracorporeal methods for the removal of LDL-Chave involved two separate steps. First, the blood must be separatedinto cellular components and plasma components. This is usually donethrough centrifugation or filtration. Second, the plasma is treated toremove LDL-C. Finally, the treated plasma and cellular components arereturned to the patient. The procedures are both time consuming andrequire a great deal of handling of blood products, which leads toincreased potential for infections. Methods involving a closed systemwhich are relatively rapid, efficient and require limited handling ofblood are highly desirable.

SUMMARY OF THE INVENTION

The present invention provides high efficiency removal of low densitylipoprotein cholesterol complex (LDL-C) directly from whole blood usingan immobilized affinity agent on a microporous plasmapheresis membrane.LDL-C removal is achieved during the plasmapheresis process. Theimmobilized affinity agent is polyacrylic acid bound directly and/orthrough an interaction with silica and/or calcium chloride to amicroporous polysulfone hollow fiber membrane.

In one aspect, the invention relates to the effective and highlyspecific removal of LDL-C from the plasma portion of whole blood. Theinvention removes negligible amounts of HDL or other blood proteins.

In another aspect the invention is superior to prior extracorporealmethods in that whole blood passes through one device where it issimultaneously separated into plasma and cellular components, the LDL-Cis removed from the plasma, and the treated plasma and cellularcomponents are returned to the patient. The process is approximatelytwice as rapid as conventional treatments and requires a minimum amountof blood handling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram indicating the action of the device of theinvention.

FIG. 2 illustrates graphically the ability of the device of theinvention to lower total cholesterol and LDL-C without effecting HDL andfree hemoglobin levels.

DETAILED DESCRIPTION OF THE INVENTION

A membrane has been discovered which has properties that areadvantageous for the removal of the complex of low density lipoproteinand cholesterol (LDL-C) from whole blood or plasma. The polysulfonehollow fiber membrane has polyacrylic acid immobilized on its surface.The membrane has desirable mechanical and specificity characteristicsfor its intended purpose of LDL-C removal. The membrane can also besterilized by autoclaving techniques.

The Membrane

The membranes of this invention are polysulfone-based polymericcompositions. Polysulfones are a known I5 class of polymers which havebeen used to form various types of membranes. Polysulfone membranes areof a substantially non-flexible physical form. "Polysulfone","polyarylsulfone", "polyether sulfone", and "polyarylether sulfone" areeach intended to define a polymeric material having a combination ofsulfone groups, aryl groups, and ether groups in the polymer chain andwhich may also contain alkylene groups therein. Polysulfone (PS)polymers are available in a variety of grades with respect to molecularweight, additives, etc. High molecular weight polysulfones may bepreferred for preparation of membranes with additional strength. Udel™P-1700, and Udel™ 3500 polysulfone polymers (Amoco Performance ProductsInc.) are suitable. Other suitable commercially available polysulfonesare under the tradenames of Astrel (3M), Victrex (ICI), and Radel(Amoco). Polysulfone is used as the primary polymeric component of themembrane because of such beneficial characteristics as thermalstability, resistance to acid, alikali and salt solutions, highmechanical strength, etc.

The polysulfones found useful as membrane components of the presentinvention are polyandry ether sulfones. the polysulfone can be viewed ashaving recurring units which is shown below: ##STR1## where the SO₂group may be in the ortho, meta or para position on the ring and where Rrepresents ##STR2## wherein n is an integer of 0 to 3 (preferably 0or 1) and each R' independently is selected from hydrogen or a C₁ -C₃alkyl, preferably methyl. The above polyarylether sulfones may be usedas homopolymers or as copolymers of the polymeric groups described abovewhere R is selected from more than one of the groups describedhereinabove. Further, the above polyarylether sulfones may be formedinto copolymers with polysulfone groups which are void of ether groupstherein such as: ##STR3## and the like. The homopolymers and copolymersdescribed above can e used as the sole polymeric component or mixturesor blends of the homopolymers and/or copolymers can be sued as themembrane component. The formation of blends provides polymeric componentwhich can have customized properties. For example, it is known thatincrease in ether oxygen and/or alkylene groups in the subject polymersprovides decrease in the soften temperature of the polymeric componentand, therefore, aids in providing a composition which can be processedat a designed temperature. The subject polysulfones can be prepared byknown manners.

The polysulfones used herein should have a weight average molecularweight of from about 20,000 to about 200,000, preferably at least about50,000 to about 150,000. The polymer Tg will be dependent upon thestructure of the polymer as described above and can be determined by oneskilled in the art by conventional analytical means.

The subject polysulfones have benzylic hydrogens which can beindependently substituted by non-dissociative groups, such as alkyl(preferably C₁ -C₃ alkyl) or halogen (preferably chlorine) or by adissociative group, such as sulfonic or carboxylic acid group. Each ofthe aryl groups may be unsubstituted or substituted with one or more ofparticular groups described above or may be substituted by differentgroups on a single aryl group or each on different aryl groups.

Other polymers or prepolymers can be used in combination with thepolysulfone polymer, if desired, to impart various characteristics tothe membrane product. Polyethylene glycol (PEG), polyvinyl pyrrolidone(PVP) or any of a variety of polyurethane prepolyers may be used withthe polysulfone to prepare these membranes. Polymers or prepolymers areadded to the polysulfone polymer in order to modify the structure andsurface additional polymer or prepolymer becomes an integral part of themembrane structure.

A. The Casting Solution

The casting solution is a multicomponent solution comprising polymericand solvent components. The primary polymeric component will be thepolysulfone polymer. The polymeric component would, of course, alsocomprise any other polymer or prepolymer which is used together with thePS polymer to form the membranes. Where reference is made to thepolysulfone solution or casting solution, it is intended to include allpolymeric components. That is, it will include the polysulfone polymerand, where appropriate, it also will include a selected additionalpolymer or prepolymer as described above.

The solvent component of the casting solution must be one in whichpolysulfone (as well as any other polymer or prepolymer used) issoluble. The polysulfone polymer is soluble in various solvents, such as4-butyrolactone, N-methylpyrrolidone (N-MP), dimethylformamide (DMF),N,N-dimethylacetamide (DMA), cyclohexanone, and chloroform.4-Butyrolactone is the preferred solvent.

At least about 8.0 wt.% and up to about 35.0 wt.% polysulfone in solventshould be used, preferably about 8.0 to about 22.0 wt.%. Above 35 wt. %,it will be difficult or impossible to dissolve the polysulfone in thesolvent. Below about 8%, precipitation will be too slow for formation ofhollow fibers, and the fibers are too fragile to handle practically. Upto about 20.0 wt.% of a second polymeric component, that is, one or moreof the polymers or prepolymers described above, can be added to the PSsolution.

The casting solution can also contain silica. Silica can be present inamounts of about 0.1 to about 10% wt/wt, preferably about 5%. The silicaaids in the immobilization of polyacrylic acid to the membrane duringthe next step of processing. Silica acts as a pore former andviscosifier to achieve a microporous structure with a nominal pore sizeof about 0.4 micron to about 0.65 micron. The casting solution can alsocontain polyacrylic acid (PAA). PAA can be present in amounts of about0.01 to about 2% wt/wt, preferably about 0.5-1%.

B. Precipitation Solution

The precipitation or coagulation mechanism of membrane formation isaffected by the composition of the precipitation solution as well asthat of the casting solution, and the composition of these two solutionsare interdependent. In this disclosure, the terms "precipitationsolution", "coagulation solution," "quench solution," and "quench bath"are used interchangeably to refer to the solution in which the membraneis formed. For formation of hollow fiber membranes, both an outer and acenter precipitation or quench solution will be employed. The solventcontent of the precipitation solution controls the rate at which thesolvent comes out of the casting solution. In turn, this controls therate of increase of the polymer concentration to the point at which thepolymeric component precipitates out of the casting solution to form themembrane. The same solvent usually is used in the casting solution andthe precipitation solution. 4-butyrolactone and blends of4-butyrolactone and N-methylpyrrolidone are the preferred solvents.Other solvents are discussed above with regard to casting solutions.

A non-solvent is often used in the precipitation solution in order toprecipitate the polymer from the casting solution, thus causingformation of the membrane. For practical and economical purposes, it ispreferred to use water as the non-solvent component of the precipitationsolution. However, other non-solvents such as methanol, ethanol,propanol, butanol, ethylene glycol, acetone, methyl ethyl ketone, or thelike, can be used instead of water, particularly when the solvent iswater-immiscible. Alternatively, water and one or more othernon-solvents can be used together.

In utilizing the method of this invention to prepare hollow fibermembranes, the precipitation solution used for the outer quench bath maybe different from that used for the center quench fluid. In thepreferred embodiment of this invention, the outer precipitation solutionis water, and the center precipitation solution is 4-butyrolactone.Other solvents and non-solvents can be used as described above. Inhollow fiber production, the center quench and outer quench aredifferent phenomena. At center quench, a small volume of solution isused, which is almost in a static mode as compared with the castingsolution. Conversely, the outer quench bath is present in large volumesand in a dynamic mode.

C. The Hollow Fiber Soinnino Conditions

In preparing the hollow fiber membranes of this invention, aliquid-liquid or wet spinning process is used similar to that describedin U.S. Pat. No. 4,970,030. That is, the casting solution is fed throughan extrusion die (spinnerette) directly into a precipitation bath, whilesimultaneously introducing the center quench fluid through the centralaperture of the spinnerette to mechanically maintain the hollow centerhole of the fiber. The fiber is fabricated and simultaneously quenchedas it is drawn through the precipitation bath. By using thiswet-spinning process, fibers with homogeneous pore structure andmembrane morphology are produced.

One of the key factors in preparation of the hollow fiber membranes ofthis invention is use of the wet spinning process; that is, spinning thecasting solution under water. In addition, selection of appropriatesolutions for the inner and outer precipitation baths is important, asis the appropriate drawing or spinning rate of the fiber as it isformed. The presence of the center quench fluid also allows forsimultaneous polymer precipitation from both the inner and outersurfaces of the fiber. The spinning rate is adjusted to allow forexchange of components between the casting and precipitation solutions.The solvent is leached out of the casting solution and is replaced bythe non-solvent from the precipitation solution. As a consequence,polymer precipitation occurs, leading to formation of the membrane.

Too rapid a drawing rate will cause breakage due to insufficientmembrane formation to maintain membrane integrity or will causeelongation or deformation of the pores. Conversely, too slow a drawingrate will cause defects resulting from excessive pressure by the centerquench solution, which may cause blow-outs in the fiber structure; also,non-circular fibers are produced. The preferred drawing rate will dependin part on the casting solution viscosity and temperature and in part onthe factors described below. However, the drawing rate typically will bein the range of about 3.0 to about 30.0 feet per minute, preferablyabout 7.0 to about 15.0 feet per minute, and will produce round fibers.

The precise spinning conditions are adjusted in order to yield hollowfibers meeting the desired physical requirements of inner diameter andwall thickness. Centering of the central aperture of the spinnette isrequired in order to achieve a fiber having a uniform wall thickness.Any spinnerette suitable for the preparation of hollow fiber membranesmay be used to prepare the membranes of this invention, however, quartzor glass spinnerettes are preferred in order to achieve the small insidediameters required of the hollow fibers of the invention. The spinningconditions left to be adjusted are the flow rate and pressure of thecasting solution and the flow rate and pressure of the center quenchfluid. These adjustments are well within the knowledge and ability ofone of ordinary skill in this art. The preferred temperature for thecasting solution will be in the range of ambient temperatures, althoughhigher temperatures, e.g., up to about 70° C., may be employed to reducethe viscosity of the casting solution.

The dimensional and porosity characteristics of the membranes of thisinvention are such that LDL-C can pass through the fiber wall but mostblood cells do not. Hemolysis occurs if numerous blood cells passthrough the fibers, which is highly undesirable. However, passage of asmall number of red blood cells through the fiber is acceptable.Generally speaking, membranes can be prepared which possess a porediameter of between about 0.1 microns to about 0.7 microns, preferablybetween 0.4 and 0.65 microns. The inner diameter of the hollow fiberscan range from about 150 to about 400 microns, preferably about 325microns. The wall thickness can range from about ten to several hundredmicrons, preferably about 75 to about 100 microns.

D. Polvacrvlic Acid Immobilization

Polyacrylic acid (PAA) is a semi-selective affinity agent for LDL-C. Thepresence of PAA on the surface of the PS hollow fiber membrane enablesthe effective removal of LDL-C from the plasma components of wholeblood. Polyacrylic acid is immobilized on the surface of the fiber wallswhen the fibers are autoclaved for about 20 to about 40 minutes at about120° to about 130° C. in a solution containing a caustic and PAA. PAA ispresent in amounts of about 0.01 to about 2% wt/wt, preferably about0.5-1%. The caustic can be 0.3 M to 1.5 M sodium hydroxide, preferably1.0 M sodium hydroxide. This is a very simple and inexpensive means foranchoring PAA onto the surface of porous membranes for use as anaffinity agent to effectively bind LDL-C. Without wishing to be bound byany theory, it is believed that the PAA is immobilized only on the outersurface of the PS hollow fiber membrane by inter-penetrating network(IPN).

PAA can be immobilized during the autoclaving step directly to the PShollow fiber membrane or it can be immobilized indirectly throughinteractions with silica which can be added to the casting solution andembedded in the PS hollow fiber membrane. Greater amounts of PAA areimmobilized to the membrane when silica is incorporated than without.While the actual nature of the interaction between PAA and silica isunknown, it is clear that silica enhances the quantity of PAA bound tothe membrane. Prior to this invention, this enhancement bulk of thesilica is removed. This step also causes the fibers to be annealed andremain unaffected by subsequent autoclave steps. Fibers with silica arenot microporous until the fibers are autoclaved in the base to removethe bulk of the silica.

Calcium chloride can also be added in or prior to this first autoclavingstep to increase again the amount of PAA immobilized to the membrane,presumably by increasing the number of binding sites. While the actualnature of the interaction between PAA and calcium chloride is unknown,it is clear that calcium chloride enhances the quantity of PAA bound tothe membrane. Calcium chloride is added to the first autoclave solutionin an amount of 0.01 to 3% wt/wt, preferably about 1%.

E. Sterilization/Cleaning

The membrane of the invention is treated in a manner to ensure that itis sterile, the fibers are annealed, and also that no trace of residualsolvent is present in the final membrane to reduce any chance of solventor unsterile products leaching into the patient. Forsterilization/cleaning the membrane is autoclaved a second time forabout 20 to about 40 minutes at 120° to 130° C. in deionized water. Themembrane is washed again in water and soaked overnight in a water bathat ambient temperature containing about 5 to about 20% glycerine. Thissterilization/cleaning process removes residual amounts of solvent andnon-immobilized PAA. Unbound calcium chloride is removed by chelation.It is important that all calcium chloride is bound or removed bychelation to ensure that the membrane is not hemolytic and does notcause complement activation.

It is important to note that, if the fibers are autoclaved first inwater, then in PAA, calcium chloride, and base, no PAA is incorporatedin the membrane.

The Device

The membranes are dried, preferably at room temperature in aircontaining less than 50% relative humidity to remove excess water. Thefibers are then placed in a housing, and both ends of the fiber arepotted in place in the housing. The preferred housing is a Focus™70housing (National Medical Care, a division of W. R. Grace & Co.-Conn.)which is packed to about 42% packing density with about 1,200 fibers perhousing. Other convenient hollow fiber housings may be used.

Use

The membranes and the device of this invention are excellently suitedfor removal of LDL-C from whole blood or plasma. FIG. 1 is a schematicrepresentation of the mechanics involved in using the LDL-C removaldevice of the invention. Whole blood is removed from the patient,typically from a vascular access point in arm 10 using suitable bloodremoval apparatus 14. Some suitable apparatus for blood removal includehypodermic needles, fistulas, subclavian catheters or other in-dwellingcatheters. The blood passes from blood removal apparatus 14 into wholeblood tubing 16 and is pumped via optional blood pump 18 into LDL-Cremoval device 28. As whole blood is pumped through the lumen of thehollow fiber membrane of LDL-C removal device 28, plasma is forcedthrough the channels of the microporous fibers and separated from thecellular components of the blood. The plasma is treated in LDL-C removaldevice 28 exiting via plasma exit port 30. The remaining bloodcomponents (high hematocrit blood) passes down through the lumen of themembrane(s) and out exit port 34. The treated plasma is pumped viaoptional plasma pump 32 through plasma tubing 36 and is reunited withthe high hemocrit blood at junction 44. The whole blood is then returnedto the patient along with additional saline 38 added through salinetubing 40 at junction 46 as necessary via return tubing 42 to suitableblood return apparatus 12. The pressure is monitored by monitor 20before blood enters LDL-C removal device 28, while blood is in LDL-Cremoval device 28 by monitor 24 and as blood exits LDL-C removal device28 by monitor 22. Pressure can be adjusted as necessary using blood pump18.

Within the LDL-C removal device the action is as follows. The nominalpore size of the hollow fiber is such that it will reject or prevent thepassage of blood cells through the membrane, yet permits the freepassage of plasma and specifically the high molecular weight componentssuch as LDL-C (2-6 million Daltons) through the membrane wall structure.As the plasma passes through the wall of the membrane, it comes intodirect contact with the affinity agent PAA, and LDL-C is bound to thewall surface. The plasma which exits through the outer surface of themembrane contains less LDL-C. In a single step, the hollow fibercartridge separates the plasma from the blood, removes the LDL-C fromthe plasma, and returns both plasma and blood components to thepatients. Under normal operating condition (flow rate (Q)_(Plasma)≦.2Q_(inlet) and transmembrane pressure (TMP)<40 mm Hg), the cartridgeis saturated with LDL-C in about 30-40 minutes. The cartridge can besubstantially regenerated with a 1.0M salt wash. This substantialregeneration represents about 85-95% of the original binding capacityrestored.

In many of the devices of the prior art, an arterial/venous fistula mustbe implanted in the patient prior to treatment in order to achieve bloodaccess to support the required higher flow rates for the devices. Theaccess is often in the form of a subclavian catheter and the implantprocedure is very invasive. The implant procedure carries certain riskswith it as well, such as increased chance of blood clots. The device ofthe present invention does not require such high flow rates, andtherefore conventional direct intravenous therapy type vascular accessis possible. This procedure is much less invasive and has fewer risksassociated with it. The flow rates of the device of this invention areoptimal when plasma outlet flow is maintained at equal to or less than20% of the blood inlet flow rate and when the pressure differencebetween the blood inlet and plasma outlet (TMP) is maintained at less orequal to 40 mm Hg. Back pressure is maintained on the plasma outlet flowto prevent the molysis in accordance with standard procedures forplasmapheresis membranes.

The membranes and device of this invention dramatically reduce theamount of LDL-C from whole blood or plasma. A significant quickreduction in LDL-C levels is advantageous for some patients and cannotbe obtained using drug or dietary regimens. The present device alsodrops LDL-C levels very selectively and effectively which is notnecessarily the case for prior art devices. The invention further canfacilitate plaque regression of atherosclerotic lesions insofar asreduction of circulating LDL-C levels permits.

This device is useful for reducing LDL-C in any number of increasedcholesterol disorders. The primary candidates for use of the device ofthe invention include young individuals homozygous for familialhypercholesterolemia who have a family history of heart disease,patients with severe coronary artery disease that are non-operable, andall potential bypass candidates. The most significant and acutecholesterol disorder is hypercholesterolemia and treatment of thisdisorder is certainly applicable to the device of the invention.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention. The following abbreviations have been used throughout indescribing the invention.

    ______________________________________                                        dl        deciliter(s)                                                        °C.                                                                              degrees centrigrade                                                 Q         flow rate                                                           g         gram(s)                                                             HDL       high density lipoprotein                                            hr        hour(s)                                                             kD        kilodalton(s)                                                       l         liter(s)                                                            LDL-C     low density lipoprotein cholestrol complex                          m         meter(s)                                                            ml        milliliter(s)                                                       min       minute(s)                                                           M         molar                                                               %         percent                                                             PAA       polyacrylic acid                                                    PS        polysulfone                                                         psi       pounds per square inch                                              rpm       rotations per minute                                                T.C.      total cholesterol                                                   TMP       transmembrane pressure                                              ______________________________________                                    

Example 1

A particular membrane of the invention having polyacrylic acid andsilica bound to the polysulfone hollow fiber membrane is prepared asfollows. Polysulfone, 210 g (Udell 1700, CAS #25135-51-7), was added to1690 g of 4-butyrolactone (Kodak, CAS #96-48-0), in a glass jar with asealable top containing a teflon (or other inert) liner. The mixture wasrolled continuously on a roller mill for 48-72 hours at room temperatureuntil the polymer was dissolved. To this solution of polysulfone in4-butyrolactone was added 100 g of silica (Sylox ™2, Davison Division ofW. R. Grace & Co.-Conn.). The jar was resealed and rolled continuouslyon the roller mill for at least 16 hours at room temperature to dispersethe silica particles. This gave a casting solution that was 10.5 wt% inPolysulfone, 5 wt% in Sylox-2 and 84.5 wt% in 4-butyrolactone.

The casting solution was then centrifuged at 2,000 rpm for 10 minutes tosettle any poorly suspended silica particles. Next, the casting solutionwas pumped through a 40 micron stainless steel screen at 60 psi ofpressure with dry nitrogen gas as the source of the driving pressure.After filtration the casting solution was de-gassed under mechanicalvacuum at less than 10 mm Hg for at least 15 minutes and put in astainless steel kettle that could be pressurized for delivery of castingsuspensions to nozzle. No substantial solvent was lost during thisdegassing procedure due to the low volatility of the solvent. Under 60psi of dry nitrogen gas, the casting solution was extruded through aglass nozzle within an orifice under the surface of a bath of deionizedwater. The core liquid of the spinnerette was 4-butyrolactone, driven by40 psi dry nitrogen gas. The hollow fiber fabricated from the processduring the under water spinning process was collected on a revolvingwheel partially submerged under water. When the appropriate number offibers were collected (800-1,200 revolutions), the fiber bundle wasremoved from the wheel, cut to chosen lengths, and soaked 16 hours atroom temperature in deionized water.

Polyacrylic acid (CAS #9003-01-4) was immobilized on the fibers in thefollowing process. Three (3) bundles of fibers of 8-inch length, eachcontaining 800 fibers, were placed in 400 ml of a solution of 1.0 Nsodium hydroxide solution containing 1% polyacrylic acid (Aldrich,M.W.=250,000) in a stainless steel tray. The fibers in this solutionwere placed in an autoclave and heated under pressure at 130° C. at 30psi for at least 30 minutes. The fibers were then removed from theNaOH/PAA solution and placed in 400 ml of deionized water and autoclavedagain for 30 minutes at 130° C. at 30 psi of deionized water. The fiberswere then soaked 16 hours at room temperature in a water bath containing20% glycerine. After soaking in the glycerine bath the fibers wereremoved and allowed to air dry for 24 hours at room temperature. Thedried fiber bundles were placed in the proper size device, and both endswere potted in place with a biomedical grade epoxy-resin system (Emerson& Cummings, Division of W. R. Grace & Co.-Conn., Cat #674A and 674B) asper instructions. The fiber device was now ready for testing after theexcess fiber and potting compounds were trimmed from both ends. Once thedevice was tested to ensure the microporous membranes maintainedpressure as expected, it was ready to be used for removal of LDL-C fromplasma and/or whole blood.

Example 2

Another membrane of the invention was prepared as described in Example 1with the following differences. The casting solution contained 10.25%Polysulfone instead of 10 5%. The first autoclave step contained 0.5%polyacrylic acid and 0.8% calcium chloride.

Example 3

A hollow fiber device containing 400 fibers as prepared in Example 1with a surface area of 226.0 cm² and total wall volume of 3.0 ml. wasperfused with plasma from a 100 ml reservoir of high cholesterol humanplasma. The recirculation of high LDL-C plasma through the device wasmaintained at a flow rate of 38 ml/min giving a shear rate of 550 sec⁻¹to achieve a steady plasma filtration rate through the walls of thefibers. Plasma samples were taken from the plasma exit port andfiltrated at time 0, 30 minutes, and 60 minutes. The averagetransmembrane pressure (TMP) remained constant throughout the run at 90mmHg, with the inlet pressure 95 mmHg and the outlet pressure 80 mmHg.Plasma filtrate flux values were 15.9 1/hr/m² at 30 minutes and 21.11/hr/m² at 60 minutes. Total cholesterol assays were performed on theplasma reservoirs using standard calorimetric assays (Sigma, No. 352-50)and nephelometry (Beckman Auto ICS Catalog Number 449310) to determinethe level of the LDL-C associated protein apolipoprotein B.

The total cholesterol (T.C.) level was reduced from an initial value of321 mg/dl to 241 mg/dl. The apolipoprotein B concentration was reducedfrom 152 mg/dl to 50 mg/dl. The albumin levels, also determined on theBeckman ICS nephelometer remained unchanged at 2,600 mg/dl. Thedifference in the pre- and post-total cholesterol values was used todetermine the amount of T.C. removed from the plasma reservoir and adrop of 25.2% was observed. This corresponds to a binding of 27 mg totalcholesterol per ml. of fiber wall volume.

Example 4

A hollow fiber device containing 1,200 fibers described in Example 2with a surface area of 2,215 cm² and a total wall volume of 20.9 ml wasperfused with a 400 ml reservoir of human whole blood. The recirculationof the whole blood through the device was maintained at a flow rate of69.0 ml/min giving a shear rate of 270 sec⁻¹. Whole blood samples fromthe reservoir and plasma samples from the filtrate were taken at time 0,15 minutes, 30 minutes, and 60 minutes. The transmembrane pressure wasmaintained at 30 mmHg with the inlet filtrate flux values were 1.91/hr/m² at 60 minutes. Total cholesterol assays were performed on thesamples using standard calorimetric assays (Sigma, No. 352-50) andnephelometry (Beckman Auto ICS, Catalog Number 449310) to determine thelevel of the LDL-C associated protein apolipo-protein B.

The total cholesterol (T.C.) level was reduced from an initial value of450 mg/dl to 339 mg/dl. The apolipoprotein B concentration was reducedfrom 267 mg/dl to 146 mg/dl. The total protein level was reduced from7.1 g/dl to 6.6 g/dl. After corrections for dilutional effects,non-specific absorption, and reservoir size, the total cholesterol levelwas lowered by 24.7% which corresponds to a binding of 9.4 mgs of totalcholesterol per ml of fiber volume.

Example 5

The procedure and device described in Example 4 was used with thefollowing differences. The device as perfused with 500 ml ofreconstituted human blood. The reconstituted human blood consisted ofconcentrated plasma containing high LDL-C levels mixed with fresh humanblood cells. Whole blood samples from the reservoir and plasma samplesfrom the filtrate were taken at time 0, 30 minutes, 60 minutes, and 90minutes. Standard assay procedures were used to determine levels oftotal cholesterol, LDL-C, HDL, and free hemoglobin.

The results shown in FIG. 2 indicate that the device is capable oflowering cholesterol to acceptable ranges in a very short time withoutsignificantly effecting HDL levels. Free hemoglobin only increased 19mg/dl over the course of treatment. This result indicates that thefibers are non-hemolytic since a value of less than 25 mg/dl isconsidered non-hemolytic.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, sincethese are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art withoutdeparting from the spirit of the invention.

We claim:
 1. A membrane for binding low density lipoprotein cholesterolconsisting essentially of a microporous polysulfone hollow fibermembrane having substantially uniform pore diameters in the range ofabout 0.1 to about 0.7 microns and having an amount of polyacrylic acideffective to bind low density lipoprotein cholesterol immobilized byinter-penetrating network on the surface of said hollow fiber membrane.2. The membrane of claim 1 which is effective to remove low densitylipoprotein cholesterol from whole blood or plasma.
 3. The membrane ofclaim 1 in which the uniform pore diameter ranges from 0.4-0.65 microns.4. The membrane of claim 1 wherein the inside diameter of the hollowfiber is about 150 to about 400 microns.
 5. A membrane for binding lowdensity lipoprotein cholesterol consisting essentially of a microporouspolysulfone hollow fiber membrane having substantially uniform porediameters in he range of about 0.1 to about 0.7 microns and havingpolyacrylic acid and calcium immobilized by inner-penetrating network onthe surface of said hollow fiber membrane, wherein the amount ofpolyacrylic acid is effective to bind low density lipoproteincholesterol and the calcium is in an amount effective to enhance thequantity of polyacrylic abide immobilized on the membrane.
 6. A membranefor binding low density lipoprotein cholesterol consisting essentiallyof a microporous polysulfone hollow fiber membrane having substantiallyuniform pore diameters n the range of about 0.1 to about 0.7 microns andhaving polyacrylic acid, calcium and silica immobilized byinner-penetrating network on the surface of said hollow fiber membrane,wherein the amount of polyacrylic acid is effective to bind low densitylipoprotein cholesterol and the calcium and silica are in an amounteffective to enhance the quantity of polyacrylic acid immobilized on themembrane.